Whole genome expression profile of dsap18 mutant Drosophila melanogaster embryos

SAP18 is a highly evolutionarily conserved protein found in all eukaryotic organisms from Saccharomycces cerevisiae to humans. It has been proposed to be involved in multiple cellular processes, from gene regulation to mRNA processing, playing different cellular functions, such as cell fate control during development or regulation of apoptosis or salt stress responses. Previous studies found SAP18 capable of repressing transcription when tethered directly to a promoter region. To gain further insight into the role of Drosophila SAP18, we performed genome-wide expression profiling of a dSAP18 mutant allele and found that dSAP18 is required for the expression of several immune-related genes, amongst others. To provide an updated view of the transcriptome, the probe sequences from the platform used (GPL3797) were matched to FlyBase Genome Release 5.5 and the corresponding gene names and identifiers were obtained. In the cases where one probe matched more than one gene, all of them were obtained. A total of four microarrays were hybridized in biological replicate pairs, such that the total RNA from dsap18 mutant animals (dsap18^117/Df(3R)sbd^45 0-24h staged embryos) and control animals (rescued dsap18^117/Df(3R)sbd^45 0-24h embryos containing a dsap18-HA transgene) used as starting material came from different extractions. Both arrays from each pair were hybridized with the same amplified RNA from sample and common reference (obtained using the Amino-Allyl Messageamp II aRNA Amplification Kit from Ambion, Inc) but with dyes (Cy3 and Cy5 from Amersham, Inc) swapped to take dye-bias into account.

SAP18是一类高度进化保守的蛋白质，广泛存在于从酿酒酵母（Saccharomyces cerevisiae）到人类的所有真核生物中。已有研究表明，SAP18参与从基因调控到mRNA加工的多种细胞过程，发挥多样化的细胞功能，例如发育过程中的细胞命运调控、细胞凋亡调控及盐胁迫应答。既往研究发现，当SAP18直接锚定至启动子区域时，可发挥转录抑制作用。为深入解析果蝇SAP18的生物学功能，我们针对dSAP18突变等位基因开展了全基因组表达谱分析，结果显示dSAP18对包括多个免疫相关基因在内的诸多基因的表达至关重要。为优化转录组分析的准确性，我们将所用芯片平台（GPL3797）的探针序列与FlyBase基因组版本5.5进行比对，获取了对应的基因名称与标识符；若单个探针匹配多个基因，则收录全部匹配结果。本研究共设置4张微阵列芯片的生物学重复配对组，实验起始材料分别来自不同提取批次的dsap18突变型动物（dsap18^117/Df(3R)sbd^45 0-24h 分期胚胎）与对照动物（携带有dsap18-HA转基因的拯救型dsap18^117/Df(3R)sbd^45 0-24h胚胎）。每对芯片均采用来自同一样本与通用参照的扩增RNA（使用Ambion公司的Amino-Allyl Messageamp II aRNA扩增试剂盒制备）进行杂交，并交换了荧光染料（Amersham公司的Cy3与Cy5），以抵消染料偏倚带来的实验误差。